The goal of this project is to elucidate the mechanisms for feedback regulation of the tip growth-signaling machinery by mechanical stresses resulting from rapid and invasive growth of tip-growing cells and the roles of mechanical feedback regulation in enabling these cells to invade tissues. Fast tip growth is vital for cells to explore the environment or reach distant destinations via guided and invasive growth, e.g., fungal pathogens invade host tissue and pollen tubes (PT) travel through female tissues to deliver sperms. Fast tip growth requires efficient tip-targeted exocytosis and tightly regulated relaxation of the apical wall inflated by high internal turgor pressure (>1 mPa). Little is known about how the tip-growing cells regulate the local relaxation of the apical wall to generate forces for rapid and invasive growth while maintaining the cell wall integrity (CWI). To address this fundamental question, the PI established Arabidopsis PT as a model system. The PI discovered a signaling network that is controlled by the ROP1 Rho-family GTPase, modulates apical exocytosis, and self-regulates ROP1 via autocrine signaling to promote tip growth. Under the prior R01 grant, modeling was integrated with experimentation to uncover an exocytosis-coordinated design principle for the regulation of tip growth. ROP1-dependent exocytosis was shown to orchestrate spatial regulation of ROP1 and wall mechanics to control tip growth and guidance. A microfluidic device-based tip-chip assay was developed to study invasive growth. Acute tensile stress resulting from invasive growth was shown to activate ROP1 via the CUP1 putative mechanosensor specifically required for maintaining CWI in PT experiencing acute stress. In contrast, CUP1 homolog, ANX, is vital for in vitro CWI likely by sensing gradual tensile stress in PT grown in vitro. This renewal resubmission will test the hypothesis that CUP1 and ANX sense acute and gradual tensile stresses respectively, which feedback regulate ROP1 signaling machinery to balance growth with CWI for efficient apical and invasive growth. Aim 1 will characterize the mechanosensory functions of CUP1 and ANX by integrating genetic and biochemical studies with single molecule mechanosensing and tip-chip assays. Aim 2 will investigate how mechanosensation by CUP1 cooperates with its sensing of a RALF peptide to achieve an acute CWI response during invasive growth. Aim 3, by using iterative modeling and experimentation, will investigate how ROP1 signaling dynamically balances the wall relaxation with maintenance of CWI, and how this balance allows the tip-growing cells to invade tissues. The project will advance the concept that the exocytosis-coordinated Rho signaling overarches fast tip growth, guidance and invasive growth, and will provide unprecedented insights into the mechanisms behind invasive growth, a process essential for cancer progression and host invasion by fungal pathogens. With conserved Rho signaling underlying these processes in diverse systems, the design principles discovered will very likely inform studies of similar processes in medically relevant systems and the discovery of drugs that specifically interrupt invasive growth.